Conventional vaccines, including attenuated or inactivated pathogens, are effective in many areas but nevertheless do not impart effective protective immunity to some infectious pathogens and tumors. This requires vaccines which are effective, versatile, ready and cost-effective to produce and easy to store.
After direct intramuscular injection of plasmid DNA had been shown to result in prolonged expression of the coded genes on the cell surface (Wolff et al., 1990), DNA-based vaccines were regarded as a new promising immunization strategy. This provided an important incentive for developing vaccines based on nucleic acids. Initially, DNA-based vaccines to infectious pathogens were tested (Cox et al., 1993; Davis et al., 1993; Ulmer et al., 1993; Wang et al., 1993) but were soon however researched in more detail also in gene therapy against tumors in order to induce specific antitumor immunity (Conry et al., 1994; Conry et al., 1995a; Spooner et al., 1995; Wang et al., 1995). This strategy of tumor immunization has a number of important advantages. Vaccines based on nucleic acids are easy to prepare and relatively inexpensive. They may moreover be amplified from a small number of cells.
DNA is more stable than RNA but carries some potential safety risks such as the induction of anti-DNA antibodies (Gilkeson et al., 1995) and integration of the transgen into the host genome. This may inactivate cellular genes, cause uncontrollable long term expression of said transgen or oncogenesis and is therefore usually not applicable to tumor-associated antigens with oncogenic potential, such as, for example, erb-B2 (Bargmann et al., 1986) and p53 (Greenblatt et al., 1994). The use of RNA offers an attractive alternative in order to circumvent these potential risks.
The advantages of using RNA as a kind of reversible gene therapy include transient expression and a non-transforming character. The RNA does not need to enter the nucleus in order to be expressed transgenically and moreover cannot integrate into the host genome, thereby eliminating the risk of oncogenesis. As with DNA (Condon et al., 1996; Tang et al., 1992), injection of RNA can also induce both the cellular and humoral immune responses in vivo (Hoerr et al., 2000; Ying et al., 1999).
The immune therapy with in vitro-transcribed RNA (IVT-RNA) makes use of two different strategies both of which have been successively tested in various animal models. Either RNA is directly injected via different routes of immunization (Hoerr et al., 2000) or dendritic cells (DCs) are transfected with in vitro-transcribed RNA by means of lipofection or electroporation and administered thereafter (Heiser et al., 2000). Recently published studies demonstrated that immunization with RNA-transfected DCs induces antigen-specific cytotoxic T lymphocytes (CTL) in vitro and in vivo (Su et al., 2003; Heiser et al., 2002). A factor of central importance for optimal induction of the T cell-mediated immune responses is inter alia the dose, i.e. density of antigen presentation on the DCs. It has been attempted to stabilize IVT-RNA by various modifications in order to achieve prolonged expression of transferred IVT-RNA and thereby to increase antigen presentation on DCs. A basic requirement for translation is the presence of a 3′ poly(A) sequence, with the translation efficiency correlating with the length of poly(A) (Preiss and Hentze, 1998). The 5′ cap and 3′ poly(A) sequence synergistically activate translation in vivo (Gallie, 1991). Untranslated regions (UTRs) of globin genes are other known elements which can contribute to stabilizing RNA and increasing translation efficiency (Malone et al., 1989).
Some IVT vectors are known in the literature which are utilized in a standardized manner as template for in vitro transcription and which have been genetically modified in such a way that stabilized RNA transcripts are produced. Protocols currently described in the literature (Conry et al., 1995b; Teufel et al., 2005; Strong et al., 1997; Carralot et al., 2004; Boczkowski et al., 2000) are based on a plasmid vector with the following structure: a 5′ RNA polymerase promoter enabling RNA transcription, followed by a gene of interest which is flanked either 3′ and/or 5′ by untranslated regions (UTR), and a 3′ polyadenyl cassette containing 50-70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenyl cassette by type II restriction enzymes (recognition sequence corresponds to cleavage site). The polyadenyl cassette thus corresponds to the later poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization and extend or mask the poly(A) sequence at the 3′ end. It is not clear, whether this nonphysiological overhang affects the amount of protein produced intracellularly from such a construct.
RNA therefore seems to be particularly suitable for clinical applications. However, the utilization of RNA in gene therapy is greatly restricted especially by the short half life of RNA, in particular in the cytoplasma, resulting in low protein expression.